Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq), to profile mouse autoantibodies. This library was validated using seven genetic mouse lines across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2–/– and µMT) were used to model non-specific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor (BCR)-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate three distinct autoimmune disease models. First, serum from Lyn–/– IgD+/– mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities. Second, serum from non-obese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, enriched peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire–/– mouse sera were used to identify numerous autoantigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 (APS1) carrying recessive mutations in AIRE. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity profiling.
Elze Rackaityte, Irina Proekt, Haleigh S. Miller, Akshaya Ramesh, Jeremy F. Brooks, Andrew F. Kung, Caleigh Mandel-Brehm, David J.L. Yu, Colin R. Zamecnik, Rebecca Bair, Sara E. Vazquez, Sara Sunshine, Clare L. Abram, Clifford A. Lowell, Gabrielle Rizzuto, Michael R. Wilson, Julie Zikherman, Mark S. Anderson, Joseph L. DeRisi
The selective targeting of pathogenic T cells is a “holy grail” in the development of new therapeutics for T cell-mediated disorders including many autoimmune diseases and graft-versus-host disease. We describe the development of a CD6-targeted antibody-drug conjugate (CD6-ADC) by conjugating an inactive form of monomethyl auristatin E (MMAE), a potent mitotic toxin, onto a monoclonal antibody (mAb) against CD6, an established T cell surface marker. Even though CD6 is present on all T cells, only the activated (pathogenic) T cells vigorously divide and thus are susceptible to the anti-mitotic MMAE-mediated killing via the CD6-ADC. We found CD6-ADC selectively killed activated proliferating human T cells and antigen-specific mouse T cells in vitro. Furthermore, in vivo, whereas the CD6-ADC had no significant detrimental effect on normal T cells in naïve CD6 humanized mice, the same dose of CD6-ADC, but not the controls, efficiently treated two pre-clinical models of autoimmune uveitis and a model of graft-versus-host disease. These results provide evidence suggesting that CD6-ADC could be further developed as a novel therapeutic agent for the selective elimination of pathogenic T cells and treatment of many T cell-mediated disorders.
Lingjun Zhang, Liping Luo, Jin Y. Chen, Rupesh Singh, William M. Baldwin III, David A. Fox, Daniel J. Lindner, Daniel F. Martin, Rachel R. Caspi, Feng Lin
Variation in the preservation of β cell function in clinical trials in type 1 diabetes (T1D) has emphasized the need to define biomarkers to predict treatment response. The T1DAL trial targeted T cells with alefacept (LFA-3-Ig) and demonstrated C-peptide preservation in ~30% of new onset T1D subjects. We analyzed islet antigen reactive (IAR) CD4 T cells in PBMC samples collected prior to treatment from alefacept- and placebo-treated subjects using flow cytometry and single cell RNA-sequencing. IAR CD4 T cells at baseline had heterogenous phenotypes. Transcript profiles formed phenotypic clusters of cells along a trajectory based on increasing maturation and activation, and T cell receptor (TCR) chains showed clonal expansion. Notably, the frequency of IAR CD4 T cells with a memory phenotype and a unique transcript profile (Cluster 3) were inversely correlated with C-peptide preservation in alefacept-, but not placebo-treated subjects. Cluster 3 cells had a proinflammatory phenotype characterized by expression of the transcription factor BHLHE40 and the cytokines GM-CSF and TNF-α, and shared TCR chains with effector memory-like clusters. Our results suggest IAR CD4 T cells as a potential baseline biomarker of response to therapies targeting the CD2 pathway and warrant investigation for other T cell-related therapies.
Elisa Balmas, Janice Chen, Alex K. Hu, Hannah A. DeBerg, Mario G. Rosasco, Vivian H. Gersuk, Elisavet Serti, Cate Speake, Carla J. Greenbaum, Gerald T. Nepom, Peter S. Linsley, Karen Cerosaletti
We previously reported that treatment of mice with 6-gingerol, the most abundant phytochemical in ginger root, leads to phosphodiesterase inhibition that counteracts neutrophil hyperactivity in models of antiphospholipid syndrome (APS) and lupus. Here, we explored the extent to which oral intake of a whole-ginger extract would similarly impact neutrophils in both autoimmune mice and healthy humans. In vitro, a solubilized ginger extract was able to attenuate neutrophil extracellular trap formation (NETosis) by human neutrophils through a mechanism that was dependent upon the cyclic AMP–dependent kinase, protein kinase A. When mice with features of either APS or lupus were administered a ginger extract orally, they demonstrated reduced circulating NETs, as well as the tempering of other disease outcomes, such as large-vein thrombosis (APS) and autoantibody production (lupus). In a pilot clinical trial, which was validated in a second cohort, daily intake of a ginger supplement for 7 days by healthy volunteers boosted neutrophil cAMP, inhibited NETosis in response to disease-relevant stimuli, and reduced circulating plasma NET levels. In summary, this work demonstrates that ginger intake restrains neutrophil hyperactivity in autoimmune mouse models and that ginger consumption by healthy individuals makes their neutrophils more resistant to NETosis.
Ramadan A. Ali, Valerie C. Minarchick, Miela Zahavi, Christine E. Rysenga, Kristin A. Sturm, Claire K. Hoy, Cyrus Sarosh, Jason S. Knight, M. Kristen Demoruelle
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with a dramatic sex bias, affecting 9-times more women than men. Activation of toll-like receptor 7 (TLR7) by self-RNA is a central pathogenic process leading to aberrant production of type-I interferon (IFN) in SLE, but the specific RNA molecules that serve as TLR7 ligands have not been defined. We therefore sought to identify female-specific endogenous RNAs containing canonical TLR7 stimulatory motifs. By leveraging gene expression data and the known sequence specificity of TLR7, we identified the female-specific X-inactive specific transcript (XIST) long non-coding RNA as a uniquely rich source of TLR7 ligands in SLE. XIST RNA stimulated IFNα production by plasmacytoid DCs in a TLR7-dependent manner, and deletion of XIST diminished the ability of whole cellular RNA to activate TLR7. XIST levels were elevated in blood leukocytes from female SLE patients compared to controls, correlated positively with disease activity and the IFN signature, and were enriched in extracellular vesicles released from dying cells in vitro. Importantly, XIST was not IFN-inducible, suggesting that XIST is a driver, rather than a consequence of IFN in SLE. Our work suggests a novel role for XIST RNA as a female-specific danger signal underlying the sex bias in SLE.
Jonathan D. Crawford, Hong Wang, Daniela Trejo-Zambrano, Raffaello Cimbro, C. Conover Talbot Jr., Mekha A. Thomas, Ashley M. Curran, Alexander A. Girgis, John T. Schroeder, Andrea Fava, Daniel W. Goldman, Michelle Petri, Antony Rosen, Brendan Antiochos, Erika Darrah
Abnormal macrophage polarization is generally present in autoimmune diseases. Overwhelming M1 macrophage activation promotes the continuous progression of inflammation, which is one of the vital reasons for the development of autoimmune diseases. However, the underlying mechanism is still unclear. Here we explore the function of RFX1 in macrophage polarization by constructing colitis and lupus-like mouse models. Both in vivo and in vitro experiments confirmed that RFX1 can promote M1 and inhibit M2 macrophage polarization. Besides, we also found that RFX1 promoted DNA demethylation of macrophage polarization-related genes by increasing APOBEC3A/Apobec3 expression. Noteworthily, we identified a potential RFX1 inhibitor, adenosine diphosphate (ADP), providing a potential strategy for treating autoimmune diseases.
Shuang Yang, Pei Du, Haobo Cui, Meiling Zheng, Wei He, Xiaofei Gao, Zhi Hu, Sujie Jia, Qianjin Lu, Ming Zhao
Many autoimmune diseases (AIDs) are characterized by persistence of autoreactive B cell responses which is often directly implicated in disease pathogenesis. How and why these cells are generated or how they are maintained for years is largely unknown. Rheumatoid arthritis is among the most common AIDs and characterized by autoantibodies recognizing proteins with post-translational modifications (PTMs). This PTM-directed, autoreactive B cell compartment is ill defined. Here, we visualized the B cell response against the three main types of PTM antigens implicated in RA by spectral flow cytometry. Our results showed extensive cross-reactivity of PTM-directed B cells against all three PTM antigens (citrulline, homocitrulline and acetyllysine). Unsupervised clustering revealed several distinct memory B cell (mBC) populations. PTM-directed cells clustered with the most recently activated, class-switched mBC phenotype, expressing high CD80, low CD24 and low CD21. Notably, patients also harbored large fractions of PTM-directed plasmablasts (PB). Both PTM-directed mBC and PB showed high expression of CXCR3, a receptor for chemokines abundantly present in arthritic joints. Together, our data provide detailed insight into the biology of B cell autoreactivity and its remarkable, seemingly exhaustless persistence in a prominent human AID.
Sanne Reijm, Joanneke C. Kwekkeboom, Nienke J. Blomberg, Jolien Suurmond, Diane Van der Woude, Rene E.M. Toes, Hans Ulrich Scherer
OBJECTIVES. Sjögren's Disease (SjD) is a chronic and systemic autoimmune disease characterized by lymphocytic infiltration and the development of dry eyes and dry mouth resulting from the secretory dysfunction of the exocrine glands. SARS-CoV-2 may trigger the development or progression of autoimmune diseases, as evidenced by increased autoantibodies in patients and the presentation of cardinal symptoms of SjD. The objective of the study was to determine whether SARS-CoV-2 induces the signature clinical symptoms of SjD. METHODS. The ACE2-transgenic mice were infected with SARS-CoV-2; SjD profiling was conducted. COVID-19 patients' sera were examined to detect the presence of autoantibodies. Clinical evaluations of convalescent COVID-19 subjects, including minor salivary gland (MSG) biopsies, were collected. Lastly, monoclonal antibodies generated from single B cells of patients were interrogated for ACE2/spike inhibition and nuclear antigens. RESULTS. Mice infected with the virus showed a decreased saliva flow rate, elevated antinuclear antibodies (ANAs) with anti-SSB/La, and lymphocyte infiltration in the lacrimal and salivary glands. Sera of COVID-19 patients showed an increase in ANA, anti-SSA/Ro52, and anti-SSB/La. The male patients showed elevated levels of anti-SSA/Ro52 compared to female patients, and female patients had more diverse ANA patterns. Minor salivary gland biopsies of convalescent COVID-19 subjects showed focal lymphocytic infiltrates in four of six subjects, and 2 of 6 subjects had focus scores >2. Lastly, we found that monoclonal antibodies produced in recovered patients can block ACE2/spike interaction and recognize nuclear antigens. CONCLUSION. Overall, our study shows a direct association between SARS-CoV-2 and SjD. Hallmark features of SjD salivary glands were histologically indistinguishable from convalescent COVID-19 subjects.The results potentially implicate that SARS-CoV-2 could be an environmental trigger for SjD.
Yiran Shen, Alexandria Voigt, Laura Goranova, Mehdi A. Abed, David E. Kleiner, Jose O. Maldonado, Margaret Beach, Eileen Pelayo, John A. Chiorini, William F. Craft, David A. Ostrov, Vijay Ramiya, Sukesh Sukumaran, Ashley N. Brown, Kaley C. Hanrahan, Apichai Tuanyok, Blake M. Warner, Cuong Q. Nguyen
The endosomal Toll-like receptor 7 (TLR7) is a major driver of murine and human systemic lupus erythematosus (SLE). The role of TLR7 in lupus pathogenesis is enhanced when the regulatory role of TLR9 is absent. TLR7 signaling in plasmacytoid DCs (pDC) is generally thought to be a major driver of the IFN response and disease pathology; however, the cell types in which TLR7 acts to mediate disease have not been distinguished. To address this, we selectively deleted TLR7 in either CD11c+ cells or CD19+ cells; using a TLR7-floxed allele, we created on the lupus-prone MRL/lpr background, along with a BM chimera strategy. Unexpectedly, TLR7 deficiency in CD11c+ cells had no impact on disease, while TLR7 deficiency in CD19+ B cells yielded mild suppression of proteinuria and a trend toward reduced glomerular disease. However, in TLR9-deficient MRL/lpr mice with accelerated SLE, B cell–specific TLR7 deficiency greatly improved disease. These results support revision of the mechanism by which TLR7 drives lupus and highlight a cis regulatory interaction between the protective TLR9 and the pathogenic TLR7 within the B cell compartment. They suggest B cell–directed, dual TLR7 antagonism/TLR9 agonism or dual TLR7/9 antagonism as a potential future therapeutic strategy to treat SLE.
Haylee A. Cosgrove, Sebastien Gingras, Minjung Kim, Sheldon Bastacky, Jeremy S. Tilstra, Mark J. Shlomchik
Systemic lupus erythematosus (SLE) affects 1 in 537 Black women, which is >2-fold more than White women. Black patients develop the disease at a younger age, have more severe symptoms, and have a greater chance of early mortality. We used a multiomics approach to uncover ancestry-associated immune alterations in patients with SLE and healthy controls that may contribute biologically to disease disparities. Cell composition, signaling, epigenetics, and proteomics were evaluated by mass cytometry; droplet-based single-cell transcriptomics and proteomics; and bead-based multiplex soluble mediator levels in plasma. We observed altered whole blood frequencies and enhanced activity in CD8+ T cells, B cells, monocytes, and DCs in Black patients with more active disease. Epigenetic modifications in CD8+ T cells (H3K27ac) could distinguish disease activity level in Black patients and differentiate Black from White patient samples. TLR3/4/7/8/9-related gene expression was elevated in immune cells from Black patients with SLE, and TLR7/8/9 and IFN-α phospho-signaling and cytokine responses were heightened even in immune cells from healthy Black control patients compared with White individuals. TLR stimulation of healthy immune cells recapitulated the ancestry-associated SLE immunophenotypes. This multiomic resource defines ancestry-associated immune phenotypes that differ between Black and White patients with SLE, which may influence the course and severity of SLE and other diseases.
Samantha Slight-Webb, Kevin Thomas, Miles Smith, Catriona A. Wagner, Susan Macwana, Aleksandra Bylinska, Michele Donato, Mai Dvorak, Sarah E. Chang, Alex Kuo, Peggie Cheung, Laurynas Kalesinskas, Ananthakrishnan Ganesan, Denis Dermadi, Carla J. Guthridge, Wade DeJager, Christian Wright, Mariko H. Foecke, Joan T. Merrill, Eliza Chakravarty, Cristina Arriens, Holden T. Maecker, Purvesh Khatri, Paul J. Utz, Judith A. James, Joel M. Guthridge
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